Differences on the constraints guiding the evolution of regulation in these domains of life. This research proposal seeks an understanding of the roles for transcription factors in assisting initiation of gene expression by RNA polymerases (RNAPs). It specifically addresses the position and function of two transcription factors, TFB and TFE, structurally and functionally conserved from archaea to humans. The significance of understanding the position and function of archaeal transcription factors to human health rests in the likelihood that human homologs of these transcription factors have the same or very similar functions that are required for proper gene expression, and that perturbations that alter their function or the function of interacting partners can cause diseases of gene dysregulation, including cancer and congenital developmental defects. This work aims to identify the structural interactions that underlie the process of transcription initiation. We expect to gain novel insight into the fine-scale positioning and function of TFB and TFE. In particular, we will have enough information about the positioning of the B-finger to create models for its function in the dynamic process of initiation, and we will also be able to test the effects of specific mutations in the B-finger using these models. We will also gain unprecedented insight into the orientation of the TFE polypeptide in transcription complexes, and will be able to identify putative interacting surfaces that can also be mutagenized and tested to elucidate function in more detail. Results from the experiments proposed here will help to drive a deeper understanding not just of the archaeal mechanism, but also of the roles for TFIIB and TFIIE (or, more specifically, the N-terminus of its alpha subunit) in transcription initiation by eukaryotic RNAP II. We also anticipate our work will help facilitate a comparison between eukaryotic-type and bacterial promoter opening mechanisms. While very different transcription factors (TFB and TFE versus sigma) assist their respective RNAPs in the process, in the end, the outcome, an elongating RNA polymerase, is geometrically the same from the point of view of the transcription bubble, the RNA-DNA hybrid, and the active site itself. When the functional similarities and differences between the two systems are understood better, we will be poised to better understand the effects of these. PUBLIC HEALTH RELEVANCE In human developmental disorders and cancers, changes in specific parts of the genome cause alterations in gene expression;as a consequence of this, cells may lose the ability to cooperate with their neighbors, and birth defects, genetic disease, or cancer can result. A detailed understanding of how these changes affect the gene expression process is incomplete, in part because the process of gene expression is not completely understood. To address this problem, I am studying the detailed mechanism of gene expression in a group of organisms called the archaea. Archaea have a transcription system that is very similar to the transcription system in human cells, though much less complicated. Therefore, they provide a relatively simple model for understanding the basic mechanisms of human gene expression.